Impact of germline DNA repair gene variants on prognosis and treatment of men with advanced prostate cancer.

The clinical importance of germline variants in DNA repair genes (DRGs) is becoming increasingly recognized, but their impact on advanced prostate cancer prognosis remains unclear. A cohort of 221 newly diagnosed metastatic castration-resistant prostate cancer (mCRPC) patients were screened for pathogenic germline variants in 114 DRGs. The primary endpoint was progression-free survival (PFS) on first-line androgen signaling inhibitor (ARSI) treatment for mCRPC. Secondary endpoints were time to mCRPC progression on initial androgen deprivation therapy (ADT) and overall survival (OS). Twenty-seven patients (12.2%) carried a germline DRG variant. DRG carrier status was independently associated with shorter PFS on first-line ARSI [HR 1.72 (1.06-2.81), P = 0.029]. At initiation of ADT, DRG carrier status was independently associated with shorter progression time to mCRPC [HR 1.56, (1.02-2.39), P = 0.04] and shorter OS [HR 1.99, (1.12-3.52), P = 0.02]. Investigating the contributions of individual germline DRG variants on PFS and OS revealed CHEK2 variants to have little effect. Furthermore, prior taxane treatment was associated with worse PFS on first-line ARSI for DRG carriers excluding CHEK2 (P = 0.0001), but not for noncarriers. In conclusion, germline DRG carrier status holds independent prognostic value for predicting advanced prostate cancer patient outcomes and may potentially inform on optimal treatment sequencing already at the hormone-sensitive stage.

Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells.

Circular RNAs (circRNA) are covalently closed molecules that can play important roles in cancer development and progression. Hundreds of differentially expressed circRNAs between tumors and adjacent normal tissues have been identified in studies using RNA sequencing or microarrays, emphasizing a strong translational potential. Most previous studies have been performed using RNA from bulk tissues and lack information on the spatial expression patterns of circRNAs. Here, we showed that the majority of differentially expressed circRNAs from bulk tissue analyses of colon tumors relative to adjacent normal tissues were surprisingly not differentially expressed when comparing cancer cells directly with normal epithelial cells. Manipulating the proliferation rates of cells grown in culture revealed that these discrepancies were explained by circRNAs accumulating to high levels in quiescent muscle cells due to their high stability; on the contrary, circRNAs were diluted to low levels in the fast-proliferating cancer cells due to their slow biogenesis rates. Thus, different subcompartments of colon tumors and adjacent normal tissues exhibited striking differences in circRNA expression patterns. Likewise, the high circRNA content in muscle cells was also a strong confounding factor in bulk analyses of circRNAs in bladder and prostate cancers. Together, these findings emphasize the limitations of using bulk tissues for studying differential circRNA expression in cancer and highlight a particular need for spatial analysis in this field of research. SIGNIFICANCE: The abundance of circRNAs varies systematically between subcompartments of solid tumors and adjacent tissues, implying that differentially expressed circRNAs discovered in bulk tissue analyses may reflect differences in cell type composition between samples.

Evaluating approaches for constructing polygenic risk scores for prostate cancer in men of African and European ancestry.

Genome-wide polygenic risk scores (GW-PRSs) have been reported to have better predictive ability than PRSs based on genome-wide significance thresholds across numerous traits. We compared the predictive ability of several GW-PRS approaches to a recently developed PRS of 269 established prostate cancer-risk variants from multi-ancestry GWASs and fine-mapping studies (PRS269). GW-PRS models were trained with a large and diverse prostate cancer GWAS of 107,247 cases and 127,006 controls that we previously used to develop the multi-ancestry PRS269. Resulting models were independently tested in 1,586 cases and 1,047 controls of African ancestry from the California Uganda Study and 8,046 cases and 191,825 controls of European ancestry from the UK Biobank and further validated in 13,643 cases and 210,214 controls of European ancestry and 6,353 cases and 53,362 controls of African ancestry from the Million Veteran Program. In the testing data, the best performing GW-PRS approach had AUCs of 0.656 (95% CI = 0.635-0.677) in African and 0.844 (95% CI = 0.840-0.848) in European ancestry men and corresponding prostate cancer ORs of 1.83 (95% CI = 1.67-2.00) and 2.19 (95% CI = 2.14-2.25), respectively, for each SD unit increase in the GW-PRS. Compared to the GW-PRS, in African and European ancestry men, the PRS269 had larger or similar AUCs (AUC = 0.679, 95% CI = 0.659-0.700 and AUC = 0.845, 95% CI = 0.841-0.849, respectively) and comparable prostate cancer ORs (OR = 2.05, 95% CI = 1.87-2.26 and OR = 2.21, 95% CI = 2.16-2.26, respectively). Findings were similar in the validation studies. This investigation suggests that current GW-PRS approaches may not improve the ability to predict prostate cancer risk compared to the PRS269 developed from multi-ancestry GWASs and fine-mapping.

Evaluating Approaches for Constructing Polygenic Risk Scores for Prostate Cancer in Men of African and European Ancestry.

Genome-wide polygenic risk scores (GW-PRS) have been reported to have better predictive ability than PRS based on genome-wide significance thresholds across numerous traits. We compared the predictive ability of several GW-PRS approaches to a recently developed PRS of 269 established prostate cancer risk variants from multi-ancestry GWAS and fine-mapping studies (PRS 269 ). GW-PRS models were trained using a large and diverse prostate cancer GWAS of 107,247 cases and 127,006 controls used to develop the multi-ancestry PRS 269 . Resulting models were independently tested in 1,586 cases and 1,047 controls of African ancestry from the California/Uganda Study and 8,046 cases and 191,825 controls of European ancestry from the UK Biobank and further validated in 13,643 cases and 210,214 controls of European ancestry and 6,353 cases and 53,362 controls of African ancestry from the Million Veteran Program. In the testing data, the best performing GW-PRS approach had AUCs of 0.656 (95% CI=0.635-0.677) in African and 0.844 (95% CI=0.840-0.848) in European ancestry men and corresponding prostate cancer OR of 1.83 (95% CI=1.67-2.00) and 2.19 (95% CI=2.14-2.25), respectively, for each SD unit increase in the GW-PRS. However, compared to the GW-PRS, in African and European ancestry men, the PRS 269 had larger or similar AUCs (AUC=0.679, 95% CI=0.659-0.700 and AUC=0.845, 95% CI=0.841-0.849, respectively) and comparable prostate cancer OR (OR=2.05, 95% CI=1.87-2.26 and OR=2.21, 95% CI=2.16-2.26, respectively). Findings were similar in the validation data. This investigation suggests that current GW-PRS approaches may not improve the ability to predict prostate cancer risk compared to the multi-ancestry PRS 269 constructed with fine-mapping.

Prognostic Value of Low-Pass Whole Genome Sequencing of Circulating Tumor DNA in Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Multiple treatments are available for metastatic castration-resistant prostate cancer (mCRPC), including androgen receptor signaling inhibitors (ARSI) enzalutamide and abiraterone, but therapy resistance remains a major clinical obstacle. We examined the clinical utility of low-pass whole-genome sequencing (LPWGS) of circulating tumor DNA (ctDNA) for prognostication in mCRPC. METHODS: A total of 200 plasma samples from 143 mCRPC patients collected at the start of first-line ARSI treatment (baseline) and at treatment termination (n = 57, matched) were analyzed by LPWGS (median: 0.50X) to access ctDNA% and copy number alteration (CNA) patterns. The best confirmed prostate specific antigen (PSA) response (≥50% decline [PSA50]), PSA progression-free survival (PFS), and overall survival (OS) were used as endpoints. For external validation, we used plasma LPWGS data from an independent cohort of 70 mCRPC patients receiving first-line ARSI. RESULTS: Baseline ctDNA% ranged from ≤3.0% to 73% (median: 6.6%) and CNA burden from 0% to 82% (median: 13.1%) in the discovery cohort. High ctDNA% and high CNA burden at baseline was associated with poor PSA50 response (P = 0.0123/0.0081), poor PFS (P < 0.0001), and poor OS (P < 0.0001). ctDNA% and CNA burden was higher at PSA progression than at baseline in 32.7% and 42.3% of the patients. High ctDNA% and high CNA burden at baseline was also associated with poor PFS and OS (P ≤ 0.0272) in the validation cohort. CONCLUSIONS: LPWGS of ctDNA provides clinically relevant information about the tumor genome in mCRPC patients. Using LPWGS data, we show that high ctDNA% and CNA burden at baseline is associated with short PFS and OS in 2 independent cohorts.

Recurrence rate after radical prostatectomy following primary staging of high-risk prostate cancer with 68Ga-PSMA PET/CT.

BACKGROUND: Accurate primary staging is one of the most important issues for initial management of prostate cancer (PCa) patients to perform an optimal selection of patients for curative intended treatment. 68Ga-Prostate-Specific-Membrane-Antigen (PSMA) PET/CT was found superior to conventional imaging both for detection of recurrence after curative intended treatment and for primary staging. We studied the recurrence rate after radical prostatectomy in high-risk PCa patients primary staged with 68Ga-PSMA PET/CT compared with conventional imaging. MATERIAL AND METHODS: The study included 247 D'Amico high-risk PCa patients treated with radical prostatectomy (RP) after primary staging with 68Ga-PSMA PET/CT and a reference group of 137 high-risk patients with RP after conventional imaging (99mTc bone scintigraphy and CT). Recurrence rates were assessed by Cox regression and Kaplan-Meier analysis. RESULTS: The 5-year recurrence-free survival rate was 71.1% in the 68Ga-PSMA PET/CT cohort compared with 56.4% in the conventional imaging cohort. Primary staging by 68Ga-PSMA PET/CT reduced biochemical recurrence (BCR) risk by 42% (HR = 0.58 (0.41-0.83), p = .004). CONCLUSION: The present data could indicate a lower recurrence rate after RP following primary staging with 68Ga-PSMA PET/CT compared to conventional imaging, likely due to improved selection of patients for surgery.

Predicting Grade group 2 or higher cancer at prostate biopsy by 4Kscore in blood and uCaP microRNA model in urine.

Elevated prostate-specific antigen (PSA) levels often lead to unnecessary and possibly harmful transrectal ultrasound guided biopsy, e.g. when the biopsy is negative or contains only low-grade insignificant cancer, unlikely to become symptomatic in the man's normal lifespan. A model based on four-kallikrein markers in blood (commercialized as 4Kscore) predicts risk of Grade group 2 or higher prostate cancer at biopsy, reducing unnecessary biopsies. We assessed whether these results extend to a single institution prostate biopsy cohort of Danish men and are enhanced by three microRNAs from urine (referred to as uCaP). The 4Kscore measured in cryopreserved blood from 234 men referred for 10+ core biopsy to Aarhus University Hospital, 29 with PSA > 25 ng/ml. We explored uCaP in urine from 157 of these men. Combined with age and DRE findings, both 4Kscore and uCaP could accurately predict Grade group 2 or higher prostate cancer (all patients: AUC = 0.802 and 0.797; PSA ≤ 25: AUC = 0.763 and 0.759). There was no additive effect when combining the 4Kscore and uCaP. Limitations include a study cohort with higher risk than commonly reported for biopsy cohorts. Our findings further support the clinical use of the 4Kscore to predict Grade group 2 or higher cancers in men being considered for biopsy.

Evaluation of Predictors of Biochemical Recurrence in Prostate Cancer Patients, as Detected by 68Ga-PSMA PET/CT.

OBJECTIVES: To explore the existence of new predictors of the 68Ga-Prostate-Specific Membrane Antigen (PSMA) PET/CT detection rate at biochemical recurrence (BCR) and to determine the detection rate of 68Ga-PSMA PET/CT dependent of prostate-specific antigen (PSA) levels. MATERIALS AND METHODS: In total, 189 PCa patients scanned with 68Ga-PSMA PET/CT for detection of BCR after curatively intended treatment with either radical prostatectomy (n = 153) or radiotherapy (n = 36) were included. Clinicopathological information at the time of diagnosis (PSA, clinical tumor-stage, International Society of Urological Pathology Grade Group and whether 68Ga-PSMA PET/CT was used for primary staging), treatment (RT/RP and histopathology of the prostatectomies), and pre-PET PSA were collected from medical records. RESULTS: Of the 189 68Ga-PSMA PET/CT scans, 103 (54.5%) were positive for BCR of PCa. No significant coherency was observed between detection rate and any clinicopathological variables at diagnosis. Detection rates significantly increased with rising PSA: <0.5 ng/mL = 28%, 0.5 ≤ 1 ng/mL = 39%, 1 ≤ 2 ng/mL = 64%, 2 ≤ 5 ng/mL = 87.5% and ≥5 ng/mL = 97%. CONCLUSIONS: The detection rate of PCa recurrence was strongly dependent of pre-PET PSA levels. None of the additional clinical variables acquired during primary staging, prostatectomy pathology reports, nor primary staging imaging modality affected the detection rate.

Independent validation of a pre-specified four-kallikrein marker model for prediction of adverse pathology and biochemical recurrence.

BACKGROUND: Accurate markers for prostate cancer (PC) risk stratification could aid decision-making for initial management strategies. The 4Kscore has an undefined role in predicting outcomes after radical prostatectomy (RP). METHODS: We included 1476 patients with 4Kscore measured prior to RP at two institutions. The 4Kscore was assessed for prediction of adverse pathology at RP and biochemical recurrence (BCR) relative to a clinical model. We pre-specified that all analyses would be assessed in biopsy Grade Group 1 (GG1) or 2 (GG2) PC patients, separately. RESULTS: The 4Kscore increased discrimination for adverse pathology in all patients (delta area under the receiver operative curve (AUC) 0.009, 95% confidence interval (CI) 0.002, 0.016; clinical model AUC 0.767), driven by GG1 (delta AUC 0.040, 95% CI 0.006, 0.073) rather than GG2 patients (delta AUC 0.005, 95% CI -0.012, 0.021). Adding 4Kscore improved prediction of BCR in all patients (delta C-index 0.014, 95% CI 0.007, 0.021; preop-BCR nomogram C-index 0.738), again with larger changes in GG1 than in GG2. CONCLUSIONS: This study validates prior investigations on the use of 4Kscore in men with biopsy-confirmed PC. Men with GG1 PC and a high 4Kscore may benefit from additional testing to guide treatment selection. Further research is warranted regarding the value of the 4Kscore in men with biopsy GG2 PC.

Immune cell analyses of the tumor microenvironment in prostate cancer highlight infiltrating regulatory T cells and macrophages as adverse prognostic factors.

Improved risk stratification is needed for patients with localized prostate cancer. This study characterized and assessed the prognostic potential of distinct immune cell infiltration patterns in the prostate tumor microenvironment. Using tissue microarrays, multiplex immunohistochemistry/immunofluorescence, and automated digital pathology, we analyzed radical prostatectomy specimens from two large patient cohorts (training: n = 470; validation: n = 333) to determine infiltration levels of seven immune cell types in malignant versus benign prostate tissue: CD3+ CD8- FoxP3- T helper cells, CD3+ CD8+ FoxP3- cytotoxic T cells (CTLs), CD3+ CD8- FoxP3+ regulatory T cells (Tregs ), CD20+ B cells, CD68+ CD163- M1 macrophages, CD68+ CD163+ M2 macrophages, and tryptase+ mast cells. Results were further validated by cell type enrichment analyses of bulk tumor RNAseq data from a third independent patient cohort (n = 99). Prognostic potential was assessed by Kaplan-Meier and uni-/multi-variate Cox regression analyses. Clinical endpoint was biochemical recurrence. All seven immune cell types were enriched in prostate cancer versus benign stroma, while there was selective enrichment for B cells, Tregs , M1 and M2 macrophages, and depletion of mast cells and CTLs in prostate cancer epithelium. In all three cohorts, high levels of infiltrating Tregs , M1, and M2 macrophages in stroma and/or epithelium were associated with biochemical recurrence (p < 0.05; log-rank test). After adjustment for routine clinical variables, Tregs and M2 macrophages remained significant adverse predictors of biochemical recurrence (p < 0.05; multivariate Cox regression). Our comprehensive analyses of immune cell infiltration patterns in the prostate tumor microenvironment highlight infiltrating Tregs , M1, and M2 macrophages as adverse predictors of prostate cancer outcome. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A polymorphism in the promoter of FRAS1 is a candidate SNP associated with metastatic prostate cancer.

BACKGROUND: Inflammation and one of its mediators, NF-kappa B (NFκB), have been implicated in prostate cancer carcinogenesis. We assessed whether germline polymorphisms associated with NFκB are associated with the risk of developing lethal disease (metastases or death from prostate cancer). METHODS: Using a Bayesian approach leveraging NFκB biology with integration of publicly available datasets we used a previously defined genome-wide functional association network specific to NFκB and lethal prostate cancer. A dense-module-searching method identified modules enriched with significant genes from a genome-wide association study (GWAS) study in a discovery data set, Physicians' Health Study and Health Professionals Follow-up Study (PHS/HPFS). The top 48 candidate single nucleotide polymorphisms (SNPs) from the dense-module-searching method were then assessed in an independent prostate cancer cohort and the one SNP reproducibly associated with lethality was tested in a third cohort. Logistic regression models evaluated the association between each SNP and lethal prostate cancer. The candidate SNP was assessed for association with lethal prostate cancer in 6 of 28 studies in the prostate cancer association group to investigate cancer associated alterations in the genome (PRACTICAL) Consortium where there was some medical record review for death ascertainment which also had SNP data from the ONCOARRAY platform. All men self-identified as Caucasian. RESULTS: The rs1910301 SNP which was reproducibly associated with lethal disease was nominally associated with lethal disease (odds ratio [OR] = 1.40; p = .02) in the discovery cohort and the minor allele was also associated with lethal disease in two independent cohorts (OR = 1.35; p = .04 and OR = 1.35; p = .07). Fixed effects meta-analysis of all three cohorts found an association: OR = 1.37 (95% confidence interval [CI]: 1.15-1.62, p = .0003). This SNP is in the promoter region of FRAS1, a gene involved in epidermal-basement membrane adhesion and is present at a higher frequency in men with African ancestry. No association was found in the subset of studies from the PRACTICAL consortium studies which had a total of 106 deaths out total of 3263 patients and a median follow-up of 4.4 years. CONCLUSIONS: Through its connection with the NFκB pathway, a candidate SNP with a higher frequency in men of African ancestry without cancer was found to be associated with lethal prostate cancer across three well-annotated independent cohorts of Caucasian men.

High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer.

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 µl of urine for small RNA sequencing in a robust and disease discriminatory manner.

FRMD6 has tumor suppressor functions in prostate cancer.

Available tools for prostate cancer (PC) prognosis are suboptimal but may be improved by better knowledge about genes driving tumor aggressiveness. Here, we identified FRMD6 (FERM domain-containing protein 6) as an aberrantly hypermethylated and significantly downregulated gene in PC. Low FRMD6 expression was associated with postoperative biochemical recurrence in two large PC patient cohorts. In overexpression and CRISPR/Cas9 knockout experiments in PC cell lines, FRMD6 inhibited viability, proliferation, cell cycle progression, colony formation, 3D spheroid growth, and tumor xenograft growth in mice. Transcriptomic, proteomic, and phospho-proteomic profiling revealed enrichment of Hippo/YAP and c-MYC signaling upon FRMD6 knockout. Connectivity Map analysis and drug repurposing experiments identified pyroxamide as a new potential therapy for FRMD6 deficient PC cells. Finally, we established orthotropic Frmd6 and Pten, or Pten only (control) knockout in the ROSA26 mouse prostate. After 12 weeks, Frmd6/Pten double knockouts presented high-grade prostatic intraepithelial neoplasia (HG-PIN) and hyperproliferation, while Pten single-knockouts developed only regular PIN lesions and displayed lower proliferation. In conclusion, FRMD6 was identified as a novel tumor suppressor gene and prognostic biomarker candidate in PC.

68Ga-PSMA PET/CT for Primary Lymph Node and Distant Metastasis NM Staging of High-Risk Prostate Cancer.

With the largest high-risk prostate cancer (PCa) cohort to date undergoing 68Ga-prostate-specific membrane antigen (PSMA) PET/CT primary staging, we aimed to, first, characterize the metastatic spread of PCa in relation to tumor 68Ga-PSMA uptake and the D'Amico classification and, second, compare 68Ga-PSMA PET/CT findings with radical prostatectomy and pelvic lymph node dissection (PLND) histopathology findings. Methods: The study included 691 consecutive newly diagnosed, biopsy-proven, treatment-naïve, D'Amico high-risk PCa patients primary-staged by 68Ga-PSMA PET/CT. PSMA SUVmax and metastatic findings were compared with prostate-specific antigen level, International Society of Urological Pathology (ISUP) grade, and clinical stage as traditional risk stratification parameters. Moreover, 68Ga-PSMA PET/CT findings were compared with histology findings in radical prostatectomy patients undergoing PLND. Undetected lymph node metastases (LNMs) underwent immunohistochemical PSMA staining. Results: Advanced disease (N1/M1) was observed in 35.3% of patients (244/691) and was associated with increasing prostate-specific antigen level, ISUP grade, and clinical stage. LNMs (N1/M1a) were detected in 31.4% (217/691) and bone metastases (M1b) in 16.8% (116/691). Advanced disease frequencies in patients with ISUP grades 2 and 3 were 10.8% (11/102) and 37.1% (33/89), respectively. Risk of advanced disease for cT2a, cT2b, and cT2c tumors was almost equal (24.2%, 27.9%, and 22.4%, respectively). We observed a weak correlation between SUVmax and biopsy ISUP grade (ρ = 0.21; P < 0.001) and a modest correlation between SUVmax and postprostatectomy ISUP grade (ρ = 0.38; P < 0.001). Sensitivity, specificity, positive and negative predictive value, and accuracy for LNM detection on 68Ga-PSMA PET/CT in the PLND cohort were 30.6%, 96.5%, 68.8%, 84.5%, and 83.1%, respectively. Undetected LNMs either were micrometastases located in the lymph node border or were without PSMA expression. Conclusion: In this high-risk PCa cohort, we identified advanced disease in about one third at diagnosis. ISUP grade was the superior predictor for advanced disease at diagnosis. We found a significant difference in frequency of advanced disease between ISUP grades 2 and 3, as supports the Gleason score 7 subdivision. Interestingly, we observed no significant differences in risk of advanced disease when comparing the different cT2 stages. The undetected LNMs were either PSMA-negative or micrometastases.

Epigenetic Analysis of Circulating Tumor DNA in Localized and Metastatic Prostate Cancer: Evaluation of Clinical Biomarker Potential.

Novel and minimally-invasive prostate cancer (PCa)-specific biomarkers are needed to improve diagnosis and risk stratification. Here, we investigated the biomarker potential in localized and de novo metastatic PCa (mPCa) of methylated circulating tumor DNA (ctDNA) in plasma. Using the Marmal-aid database and in-house datasets, we identified three top candidates specifically hypermethylated in PCa tissue: DOCK2,HAPLN3, and FBXO30 (specificity/sensitivity: 80%-100%/75-94%). These candidates were further analyzed in plasma samples from 36 healthy controls, 61 benign prostatic hyperplasia (BPH), 102 localized PCa, and 65 de novo mPCa patients using methylation-specific droplet digital PCR. Methylated ctDNA for DOCK2/HAPLN3/FBXO30 was generally not detected in healthy controls, BPH patients, nor in patients with localized PCa despite a positive signal in 98%-100% of matched radical prostatectomy tissue samples. However, ctDNA methylation of DOCK2,HAPLN3, and/or FBXO30 was detected in 61.5% (40/65) of de novo mPCa patients and markedly increased in high- compared to low-volume mPCa (89.3% (25/28) vs. 32.1% (10/31), p < 0.001). Moreover, detection of methylated ctDNA was associated with significantly shorter time to progression to metastatic castration resistant PCa, independent of tumor-volume. These results indicate that methylated ctDNA (DOCK2/HAPLN3/FBXO30) may be potentially useful for identification of hormone-naïve mPCa patients who could benefit from intensified treatment.

Profiling of Circulating microRNAs in Prostate Cancer Reveals Diagnostic Biomarker Potential.

Early detection of prostate cancer (PC) is paramount as localized disease is generally curable, while metastatic PC is generally incurable. There is a need for improved, minimally invasive biomarkers as current diagnostic tools are inaccurate, leading to extensive overtreatment while still missing some clinically significant cancers. Consequently, we profiled the expression levels of 92 selected microRNAs by RT-qPCR in plasma samples from 753 patients, representing multiple stages of PC and non-cancer controls. First, we compared plasma miRNA levels in patients with benign prostatic hyperplasia (BPH) or localized prostate cancer (LPC), versus advanced prostate cancer (APC). We identified several dysregulated microRNAs with a large overlap of 59 up/down-regulated microRNAs between BPH versus APC and LPC versus APC. Besides identifying several novel PC-associated dysregulated microRNAs in plasma, we confirmed the previously reported upregulation of miR-375 and downregulation of miR-146a-5p. Next, by randomly splitting our dataset into a training and test set, we identified and successfully validated a novel four microRNA diagnostic ratio model, termed bCaP (miR-375*miR-33a-5p/miR-16-5p*miR-409-3p). Combined in a model with prostate specific antigen (PSA), digital rectal examination status, and age, bCaP predicted the outcomes of transrectal ultrasound (TRUS)-guided biopsies (negative vs. positive) with greater accuracy than PSA alone (Training: area under the curve (AUC), model = 0.84; AUC, PSA = 0.63. Test set: AUC, model = 0.67; AUC, PSA = 0.56). It may be possible in the future to use this simple and minimally invasive bCaP test in combination with existing clinical parameters for a more accurate selection of patients for prostate biopsy.

The effect of assessing genetic risk of prostate cancer on the use of PSA tests in primary care: A cluster randomized controlled trial.

BACKGROUND: Assessing genetic lifetime risk for prostate cancer has been proposed as a means of risk stratification to identify those for whom prostate-specific antigen (PSA) testing is likely to be most valuable. This project aimed to test the effect of introducing a genetic test for lifetime risk of prostate cancer in general practice on future PSA testing. METHODS AND FINDINGS: We performed a cluster randomized controlled trial with randomization at the level of general practices (73 in each of two arms) in the Central Region (Region Midtjylland) of Denmark. In intervention practices, men were offered a genetic test (based on genotyping of 33 risk-associated single nucleotide polymorphisms) in addition to the standard PSA test that informed them about lifetime genetic risk of prostate cancer and distinguished between "normal" and "high" risk. The primary outcome was the proportion of men having a repeated PSA test within 2 years. A multilevel logistic regression model was used to test the association. After applying the exclusion criteria, 3,558 men were recruited in intervention practices, with 1,235 (34.7%) receiving the genetic test, and 4,242 men were recruited in control practices. Men with high genetic risk had a higher propensity for repeated PSA testing within 2 years than men with normal genetic risk (odds ratio [OR] = 8.94, p < 0.01). The study was conducted in routine practice and had some selection bias, which is evidenced by the relatively large proportion of younger and higher income participants taking the genetic test. CONCLUSIONS: Providing general practitioners (GPs) with access to a genetic test to assess lifetime risk of prostate cancer did not reduce the overall number of future PSA tests. However, among men who had a genetic test, knowledge of genetic risk significantly influenced future PSA testing. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov, number NCT01739062.

Epigenetic silencing of MEIS2 in prostate cancer recurrence.

BACKGROUND: Current diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, resulting in overdiagnosis and overtreatment of clinically insignificant tumors. Thus, to improve the management of PC, novel biomarkers are urgently needed. RESULTS: In this study, we integrated genome-wide methylome (Illumina 450K DNA methylation array (450K)) and RNA sequencing (RNAseq) data performed in a discovery set of 27 PC and 15 adjacent normal (AN) prostate tissue samples to identify candidate driver genes involved in PC development and/or progression. We found significant enrichment for homeobox genes among the most aberrantly methylated and transcriptionally dysregulated genes in PC. Specifically, homeobox gene MEIS2 (Myeloid Ecotropic viral Insertion Site 2) was significantly hypermethylated (p < 0.0001, Mann-Whitney test) and transcriptionally downregulated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue in our discovery sample set, which was also confirmed in an independent validation set including > 500 PC and AN tissue samples in total (TCGA cohort analyzed by 450K and RNAseq). Furthermore, in three independent radical prostatectomy (RP) cohorts (n > 700 patients in total), low MEIS2 transcriptional expression was significantly associated with poor biochemical recurrence (BCR) free survival (p = 0.0084, 0.0001, and 0.0191, respectively; log-rank test). Next, we analyzed another RP cohort consisting of > 200 PC, AN, and benign prostatic hyperplasia (BPH) samples by quantitative methylation-specific PCR (qMSP) and found that MEIS2 was significantly hypermethylated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue samples (AN and BPH) with an AUC > 0.84. Moreover, in this cohort, aberrant MEIS2 hypermethylation was significantly associated with post-operative BCR (p = 0.0068, log-rank test), which was subsequently confirmed (p = 0.0067; log-rank test) in the independent TCGA validation cohort (497 RP patients; 450K data). CONCLUSIONS: To the best of our knowledge, this is the first study to investigate, demonstrate, and independently validate a prognostic biomarker potential for MEIS2 at the transcriptional expression level and at the DNA methylation level in PC.

Elevated miR-615-3p Expression Predicts Adverse Clinical Outcome and Promotes Proliferation and Migration of Prostate Cancer Cells.

miR-615-3p has previously been described as up-regulated in prostate cancer (PC) tissue samples compared with nonmalignant controls; however, its prognostic potential and functional role in PC remain largely unknown. In this study, we investigated the clinical and biological relevance of miR-615-3p in PC. The expression of miR-615-3p was measured in PC tissue specimens from 239 men who underwent radical prostatectomy (RP), and it was investigated if miR-615-3p could predict postoperative biochemical recurrence (BCR). These findings were subsequently validated in three independent RP cohorts (n = 222, n = 273, and n = 387) and functional overexpression studies conducted in PC cells (PC3M). High miR-615-3p expression was significantly associated with BCR in four independent PC patient cohorts (P < 0.05, log-rank test). In addition, high miR-615-3p expression was a significant predictor of PC-specific survival in univariate (hazard ratio, 3.75; P < 0.001) and multivariate (hazard ratio, 2.66; P = 0.008) analysis after adjustment for the Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S) nomogram in a merged RP cohort (n = 734). Moreover, overexpression of miR-615-3p in PC cells (PC3M) significantly increased cell viability, proliferation, apoptosis, and migration. Together, our results suggest that miR-615-3p is a significant predictor of postoperative BCR and PC-specific survival and has oncogenic functions in PC cells.

Epigenetics of Urological Cancers.

The major urological cancers comprise prostate adenocarcinoma, urinary bladder (or upper urinary tract) carcinoma, renal cell carcinoma, testicular cancer and penile carcinoma, in this order of incidence, each with various histological and molecular subtypes [...].